Pharmaceutical composition comprising immunoglobulin fc-fused interleukin-7 fusion protein for preventing or treating human papillomavirus-caused diseases
a technology of interleukin-7 and fusion protein, which is applied in the direction of drug compositions, peptide/protein ingredients, organic non-active ingredients, etc., can solve the problems of wart development, precancerous lesions, and lack of therapeutic effects against pre-existing infections and hpv-induced lesions, and achieves easy-to-conduct effects
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preparation example 1
mental Animals
[0074]Female C57BL / 6 mice, 8-10 weeks of age used in the following examples were purchased from The Jackson Laboratory (Bar Harbor, USA). All animals were raised under specific pathogen-free conditions in the animal care facility in POSTECH. The procedures of animal experiments were performed in accordance with the National Institutes of Health (NIH) guidelines for mouse experiments. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Also, female Sprague-Dawley rats at 11 weeks of age were purchased from the Charles River Laboratories (Raleigh, USA). All animals were raised under specific pathogen-free conditions in the animal care facility of MPI research. The procedures of animal experiments were performed in accordance with the regulations outlined in the United States Department of Agriculture (USDA) animal welfare act (9 CFR, parts 1-3).
preparation example 2
ment of Fusion Protein of Fc and IL-7
[0075]The codon-optimized human IL-7 and granulocyte colony-stimulating factor (G-CSF) genes were individually fused with a hybrid Fc-fragment. The schematic structure of Fc-fused IL-7 is shown in FIG. 1. Chinese hamster ovary (CHO) cells were stably transfected with a plasmid encoding IL-7-Fc and G-CSF-Fc. And then, IL-7-Fc and G-CSF-Fc were obtained from the cells. Purified recombinant human IL-7 (rIL-7), for a control group, was purchased from Biolegend (San Diego, USA).
[0076]3 mg of medroxyprogesterone acetate (Depo-Provera, Pfizer) was subcutaneously injected to mice in a diestrus state 4 days before treatment. The mice were anesthetized by intraperitoneal injection with 100 mg / kg ketamine (Yuhan) and 10 mg / kg xylazine hydrochloride (Bayer) in PBS. Then, 10 μg of rIL-7, IL-7-Fc or G-CSF-Fc were mixed with PBS and applied (administered) on the vaginal mucosal tissues using a micropipette.
preparation example 3
orescence-Conjugated IL-7-Fc in the Genital Tract
[0077]IL-7-Fc was coupled with Cy-5.5 mono-reactive NHS ester. Eluted proteins were desalted and concentrated by using centrifugal filter devices (Merck Millipore) and protein concentration of the dye-labeled IL-7-Fc was measured using an anti-human IL-7 ELISA set (Southern Biotech). Cy-5.5-conjugated IL-7-Fc (1 mg / kg) and Cy-5.5 in PBS were intravaginally administered to anesthetized mice with equivalent signal intensity. At days 1 and 7 after administration, mice were euthanized and their vaginas were washed, and each of the organs was obtained. The fluorescence signal intensity was then quantified using an IVIS spectral machine (Caliper Life Science). Signal intensity was measured quantitatively in the organ by measuring photons per second per centimeter squared per steradian (p / s / cm2 / sr).
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